Multi-targets interfering RNA molecules and their applications

ABSTRACT

This invention relates to interfering RNA (iRNA) molecules and their applications, especially multi-targets iRNA molecules and their applications. The said multi-targets iRNA molecules comprised of a sense strand annealed onto at least one antisense strand, each strand is at least 30 nucleotides in length, the sense or antisense strand has at least two segments, which can target at least two RNAs of different genes, or can target at least two portions of an RNA, and wherein the iRNA does not induce an interferon-response when transfected into a cell. The iRNA molecule can interfere with the translation procedure post-transcription, and the target gene is inhibited or blocked, the iRNA does not induce an interferon-response in vivo. The RNA molecules are the active ingredient in preparation of the drug which can regulate one or many genes function.

CROSS-REFERENCE

This application is a Divisional application of U.S. patent applicationSer. No. 13/415,600, filed Mar. 8, 2012, which claims the benefit ofChinese Application Nos. 201110056947.0, 201110056948.5, and201110056949.X, all filed Mar. 10, 2011. The contents of allapplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a form of post-transcriptional gene silencingin which double-stranded RNA (dsRNA) induces the enzymatic degradationof homologous messenger RNA (mRNA). When a long dsRNA enters a cell, anenzyme called Dicer binds and cleaves the long dsRNA. Cleavage by Dicerresults in the production of a small interfering RNA (siRNA) that isgenerally 20-25 base pairs (bp) in length and has a 2-nucleotide long 3′overhang on each strand. One of the two strands of each siRNA, generallythe antisense strand, is then incorporated into an RNA-induced silencingcomplex (RISC), and pairs with complementary RNA sequences. RISC firstmediates the unwinding of the siRNA duplex, a single-stranded siRNA thatis coupled to RISC, then binds to a target mRNA in a sequence-specificmanner. The binding mediates target mRNA cleavage by Slicer, anargonaute protein that is the catalytic component of RISC. Cleavage ofthe mRNA prevents translation from occurring, which prevents theultimate expression of the gene from which the mRNA is transcribed. Now,it has been confirmed that RNAi has great potential treatment of avariety viral infections, and it is the ideal treatment for blockinggene expression.

RNAi has tremendous potential in medicinal therapeutics, such as inanti-viral, oncogenic and anti-inflammatory applications. Thedouble-stranded siRNA may be a long double-strand designed to be cleavedby Dicer, called Dicer substrate, or it may be short and is designed tobypass Dicer serve directly as a RISC substrate. The dsRNAs aresynthesized with a sequence complementary to a gene of interest andintroduced into a cell or organism, where they are recognized asexogenous genetic materials and activate the RNAi pathway. RNAi cancause a drastic decrease in the expression of a targeted gene by usingthis mechanism.

RNAi can be used to develop a whole new class of therapeutics. Currentlythere are more than ten kinds of siRNA drug at the clinic stage. Amongthe applications to reach clinical trials are treatment of age-relatedmacular degeneration, diabetic retinopathy, and respiratory syncytialvirus, solid tumors, liver cancer, and acute kidney injury and otherdiseases.

SUMMARY OF THE INVENTION

The present application provides compounds and processes related tointerfering RNA (iRNA) molecules and applications thereof. The iRNAmolecule having antisense strand that can target or hybridize to RNAs oftwo or more different target genes or two or more different sites of aRNA of a single target gene. The RNAs could be mRNA, microRNA (miRNA) ortwo or more subsequences of one mRNA or miRNA, and which does not inducean interferon response.

The present invention further provides a multiple targets iRNA having asense strand and at least one antisense strand. The length of eachstrand is at least 30, 31, 32, 33, 34, 35, 36, 40, or more nucleotides,the sense or antisense strand can target at least two different RNAs ortwo different sequences on a single RNA.

Generally, the iRNA comprises a non-complementary loop structure or aself-complementary hairpin structure, and wherein the iRNA does notinduce an interferon-response when introduced (e.g. by transfection)into a cell.

Preferably, the non-complementary loop structure contains at least 3nucleotides, and the self-complementary hairpin structure contains atleast 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides.

Preferably, the iRNA comprises a nucleotide with sugar or backbonemodification.

Usually, the iRNA comprise a sense and a antisense strand or twoantisense strands, all independently with a length that is at least 30,31, 32, 33, 34, 35, 36, 40, or more nucleotides long, and thedouble-stranded iRNA contains at least a non-complementary loopstructure and a self-complementary hairpin structure, and the iRNA doesnot induce an interferon-response when introduced into a mammalian cell.

The invention also discloses the applications of the iRNAs in the genemodulation drugs preparation, and the drug modulates at least one gene.The gene is a disease-related gene, includes pathogen gene, such asviral gene, and non-infectious disease gene, such as cancer gene andetc. The cancers is liver cancer, lung cancer, gastric carcinoma,cervical cancer, multiple myeloma, cutaneous squamous cell carcinoma,colon carcinoma, melanoma, bladder carcinoma, osteosarcoma,nasopharyngeal carcinoma, or mouth cancer, and the like.

The composition of the present invention is used to suppress, mitigateor reduce the symptoms of the disease or prevent the recurrence ofcertain diseases.

The present invention provides iRNA that can inhibit expression ofmultiple target genes, or target multiple sites of the same gene. Inaddition, it does not induce an interferon-response, can be applied tomammalian cells. The disclosed RNA molecule is a new siRNA applicationform with a broad range of applications in gene therapy for diseases.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which.

FIG. 1 shows the structure of Mid-Cir. The molecule has segment 1 of asense strand which is complementary to segment 3 of an antisense strand.Segment 2 of the sense strand is complementary to the segment 4 of theantisense strand. Mid-Cir has a non-complementary loop structure in boththe sense and the antisense strands.

FIG. 2 shows the structure of Mid-Loop. The molecule has segment 1 of asense strand which is complementary to segment 3 of an antisense strand.Segment 2 of the sense strand is complementary to segment 4 of theantisense strand. Mid-Loop has a self-complementary hairpin structure inboth the sense and the antisense strands.

FIG. 3 shows the structure of By-Pass. The molecule has a sense strandannealed onto two antisense strands, each strand has two segments (a 5′segment and a 3′ segment). The 5′ segment (segment 1) of the sensestrand is complementary to the 3′ segment (segment 3) of the firstantisense strand (antisense strand 1). The 3′ segment (segment 2) of thesense strand is complementary to the 5′ segment (segment 4) of thesecond antisense strand (antisense strand 2). The 5′ segment (segment 5)of the first antisense strand (antisense strand 1) is complementary tothe 3′ segment (segment 6) of the second antisense strand (antisensestrand 2). After annealing of the three strands, a shelf-complementaryhairpin structure is formed in the sense strand.

FIG. 4 shows the relative level of Survivin mRNA in SMMC-7721 hepatomacells transfected with various iRNA. iRNA molecules Mid-Cir, Mid-Loop,By-Pass all had shown better results than the corresponding Sur-2transfection group and Sur-2/Bcl-1 co-transfection group in Survivingene silencing.

FIG. 5 shows the relative level of Bcl-2 mRNA in SMMC-7721 hepatomacells transfected with various iRNA iRNA molecules Mid-Cir, Mid-Loop,By-Pass all had shown better results than the corresponding Bcl-1transfection group and Sur-2/Bcl-1 co-transfection group in Bcl-2 genesilencing.

FIG. 6 shows the relative level of OAS1 mRNA in SMMC-7721 hepatoma cellstransfected with various iRNA. Comparisons with the normal group, themRNA expression level of OAS1 gene in the positive control group wasincreased significantly; the OAS1 gene of other group did not have highlevel mRNA expression. The results indicated that the iRNA moleculesMid-Cir, Mid-Loop, By-Pass did not induce interferon response.

FIG. 7 shows the growth curve of SMMC-7721 hepatoma cells.

FIG. 8 shows inhibition of SMMC-7721 hepatoma cell growth by differentRNA molecules. The results show that hepatoma cells were inhibitedsignificantly after the treatment of the iRNA molecules Mid-Cir,Mid-Loop, By-Pass for 48 hrs, 72 hrs, 96 hrs.

FIG. 9 shows the growth curve of HepG2 hepatoma cells.

FIG. 10 shows inhibition of HepG2 hepatoma cell growth by different RNAmolecules. The results show that the hepatoma cells were inhibitedsignificantly after the treatment of the iRNA molecules Mid-Cir,Mid-Loop, By-Pass for 48 hrs, 72 hrs, 96 hrs.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

I. Multi-Target RNAs

In one aspect, the present invention provides compositions and methodsrelated to an iRNA for targeting RNAs of different genes or differentregion of an RNA of a single gene.

In mammalian cells, long dsRNA induces interferon response easily,therefore for application of RNAi in mammalians, the length of siRNA isgenerally be shorter than 30 bps. Nevertheless, long interfering RNA isuseful for many applications. Long interfering RNA can contain multipletargeting sequences, and thus can target multiple mRNAs of differenttarget genes or more than one site of the same mRNA of a single gene.Multi-target iRNA can increase gene silencing effect and expand therange of applications.

The development of diseases is complex biological processes with manygenes participate, and is affected by many factors. In the treatment ofdiseases single-target treatment has its limitations Inhibition of asingle gene generally cannot completely suppress or reverse thedevelopment of diseases. Thus, it is necessary to research and developlong interference nucleic acids that are double-stranded, have multipletargets, have no interferon response, and can suppress multiple diseasegenes expression. It can improve the therapeutic efficacy and be used asnew ways of treating diseases.

By “multi-target iRNA” herein is meant a RNA that targets different RNAsof different target genes or different sites of one target RNA of asingle target gene. Multi-target iRNA can be used to modulate theexpression of one or more target genes.

“Target” or “target gene” refers to a gene whose expression isselectively inhibited or “silenced.” This silencing is achieved bycleaving or translationally silencing or enhance RNA degradation of themRNA of the target gene (also referred to herein as the “target mRNA”)by an iRNA, or an RNA silencing agent, e.g., an siRNA synthesizedenzymatically or non-enzymatically, or created from an engineered RNAprecursor by a cell's RNA silencing system.

“RNA silencing agent” refers to an RNA (or analog thereof), havingsufficient sequence complementarity to a target RNA (i.e., the RNA beingdegraded) to direct RNA silencing (e.g., RNAi). An RNA silencing agenthaving a “sequence sufficiently complementary to a target RNA sequenceto direct RNA silencing” means that the RNA silencing agent has asequence sufficient to trigger the destruction or post-transcriptionalsilencing of the target RNA by the RNA silencing machinery (e.g., theRISC) or related process. An RNA silencing agent having a “sequencesufficiently complementary to a target RNA sequence to direct RNAsilencing” also means that the RNA silencing agent has a sequencesufficient to trigger the translational inhibition of the target RNA bythe RNA silencing machinery or process.

As used herein, the term multiple targets refers to the antisense strandof the interfering RNA (iRNA) targeting different RNAs of differenttarget genes or different sites of one target RNA of a single gene. TheRNA could be mRNA, microRNA (miRNA) or two or more subsequences of onemRNA or miRNA.

By “modulation” or “modulation of expression” herein is meant either anincrease (stimulation) or a decrease (inhibition) in the amount orlevels of a nucleic acid molecule encoding the gene, e.g., DNA or RNAInhibition is often the preferred form of modulation of expression andmRNA is often a preferred target nucleic acid.

Modified nucleotides in an iRNA molecule can be in the antisense strand,the sense strand, or both.

In some embodiments, iRNA molecules comprise separate sense andantisense sequences or regions, wherein the sense and antisense regionsare covalently linked by nucleotide or non-nucleotide linker molecules,or are non-covalently linked by ionic interactions, hydrogen bonding,van der Waals interactions, hydrophobic interactions, and/or stackinginteractions.

The RNA molecules can be assembled from two separate oligonucleotidesinto a duplex, where one strand is the sense strand and the other is theantisense strand, wherein the antisense and sense strands are basecomplementary. The antisense strand may comprise a nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidor a portion thereof, and the sense strand may comprise a nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof.

The use herein of the term “Nucleic acid” refers to deoxyribonucleotidesor ribonucleotides and polymers thereof in single- or double-strandedform. The term encompasses nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides.

By “RNA” herein is meant a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a beta D-ribo-furanose moiety. Theterms include double-stranded RNA, single-stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinant produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the iRNAor internally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

As used herein complementary nucleotide bases are a pair of nucleotidebases that form hydrogen bonds with each other. Adenine (A) pairs withthymine (T) or with uracil (U) in RNA, and guanine (G) pairs withcytosine (C). Complementary segments or strands of nucleic acidhybridize (join by hydrogen bonding) with each other.

As used in the invention, the term “non-complementary loop structure”refers to the non-complementary nucleotide sequence which locates in thesense strand with a corresponding sequence in the antisense strand. Thelength is at least 3 nucleotides and there is no self-complementarysequence in the nucleotide sequence. The sequence forms a looped secondstructure after the sense and antisense strand have annealed.

Chemical Composition of iRNAs

The iRNA of the present invention comprises single-stranded ordouble-stranded oligonucleotides.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure including two anti-parallel and substantiallycomplementary, as defined herein, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs. A dsRNA as used herein is alsoreferred to as an “iRNA”.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to thecorresponding segment of a highly conserved domain sequence of a viralgenome sequence. As used herein, the term “region of complementarity”refers to the region on the antisense strand that is substantiallycomplementary to a sequence, for example a target sequence, as definedherein. Where the region of complementarity is not fully complementaryto a highly conserved domain sequence, the mismatches are most toleratedin the terminal regions and, if present, are generally in a terminalregion or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand. Sense strand generally is the same strand as aRNA transcribed from a viral genome, preferably an RNA encoding aprotein.

The term “identity” is the relationship between two or morepolynucleotide sequences, as determined by comparing the sequences.Identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match between strings ofsuch sequences. While there exist a number of methods to measureidentity between two polynucleotide sequences, the term is well known toskilled artisans (see, e.g., Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); and Sequence Analysis Primer,Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York(1991)). “Substantially identical,” as used herein, means there is avery high degree of homology (e.g., 100% sequence identity) between thesense strand of the dsRNA and the corresponding part of the target gene.However, dsRNA having greater than 90% or 95% sequence identity may beused in the present invention, and thus sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence can be tolerated. Although 100% identity istypical, the dsRNA may contain single or multiple base-pair randommismatches between the RNA and the highly conserved domain sequence.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. In someembodiments, the presence of only one nucleotide overhang strengthensthe activity of the dsRNA, without affecting its overall stability.dsRNA having only one overhang has proven particularly stable andeffective in vivo, as well as in a variety of cells, cell culturemediums, blood, and serum. Generally, the single-stranded overhang islocated at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA mayalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. In one embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In one embodiment, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand. In another embodiment, one ormore of the nucleotides in the overhang is replaced with a nucleosidethiophosphate.

In some embodiments, the iRNA comprises RNA:RNA duplex with 0, 1, 2, 3,4, or 5 base pairs of DNA attached to the 3′ end or 5′ end of theRNA:RNA duplex. In some embodiments, attachment of the DNA base pairs tothe 3′end is preferred. The base pairs can have 1, 2, 3, or 4nucleotides overhang. In some embodiments, a blunt end DNA base pair ispreferred. In some embodiments, the DNA is preferably a T, or dT.

In some embodiments, the iRNA comprises 1, 2, 3, 4, or 5 bases ofdeoxyribonucleotides at the 3′-end of the nucleotide sequence. The basescan be cytosine (C), guanine (G), adenine (A), uracil (U), deoxycytidine(dC), deoxyguanosine (dG), deoxyadenine (dA), or deoxythymidine (dT), oranalogues thereof.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof.

As used herein, the term “oligonucleotide”, includes linear or circularoligomers of natural and/or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, substituted and alpha-anomericforms thereof, peptide nucleic acids (PNA), linked nucleic acids (LNA),phosphorothioate, methylphosphonate, and the like. Oligonucleotides arecapable of specifically binding to a target polynucleotide by way of aregular pattern of monomer-to-monomer interactions, such as Watson-Cricktype of base pairing, Hoogsteen or reverse Hoogsteen types of basepairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. “Chimeric oligonucleotides” or “chimeras,” in the context ofthis invention, are oligonucleotides which contain two or morechemically distinct regions, each made up of at least one nucleotide.These oligonucleotides typically contain at least one region of modifiednucleotides that confers one or more beneficial properties (such as, forexample, increased nuclease resistance, increased uptake into cells,increased binding affinity for the RNA target) and a region that is asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques.

In some embodiments, the region of the oligonucleotide which is modifiedcomprises at least one nucleotide modified at the 2′ position of thesugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other preferred embodiments, RNAmodifications include 2′-fluoro, 2′-amino and 2′ O-methyl modificationson the ribose of pyrymidines, abasic residues or an inverted base at the3′ end of the RNA. The effect of such increased affinity is to greatlyenhance iRNA oligonucleotide inhibition of gene expression. Cleavage ofthe RNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.

Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance. Some desirablemodifications can be found in De Mesmaeker et al. Acc. Chem. Res. 1995,28:366-374.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG tosynthesize fluorescently labeled, biotinylated or other modifiedoligonucleotides such as cholesterol-modified oligonucleotides.

The antisense and sense strand of the iRNA of the invention could benucleotide analogues which contain sugar or backbone modification.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc (ref:Recent advances in the medical chemistry of antisense oligonucleotide byUhlman, Current Opinions in Drug Discovery & Development 2000 Vol 3 No2). This can be achieved by substituting some of the monomers in thecurrent oligonucleotides by LNA monomers. The LNA modifiedoligonucleotide may have a size similar to the parent compound or may belarger or preferably smaller. It is preferred that such LNA-modifiedoligonucleotides contain less than about 70%, more preferably less thanabout 60%, most preferably less than about 50% LNA monomers and thattheir sizes are between about 10 and 25 nucleotides, more preferablybetween about 12 and 20 nucleotides.

Preferred modified oligonucleotide backbones comprise, but not limitedto, phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in o Nielsen et al., Science, 1991, 254, 1497-1500.

In a more preferred embodiment of the invention the oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular-CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂-known as amethylene(methylimino) or MMI backbone, —CH₂—O—N(CH₃)—CH₂—,—CH₂N(CH₃)—N(CH₃) CH₂— and —O—N(CH₃)—CH₂—CH₂— wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂— of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C to CO alkyl or C₂ to CO alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH_(2n)ON[(CH₂)_(n)CH₃)]₂ where n and m can be from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationcomprises 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification comprises 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications comprise 2′-methoxy(2′-OCH₃),2′-aminopropoxy(2′-O CH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures comprise, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514, 785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646, 265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases comprise other synthetic andnatural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopaedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., ‘AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Thesecomprise 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleobases as well as other modified nucleobasescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBSLett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Sequence of iRNAs

In some embodiments, the nucleotide sequence is at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a fragment of a target sequence.

In general, a batch of iRNAs is designed and synthesized, preferablyincludes all the possible iRNAs for a given target gene or target genes.

After one or more lead iRNAs are identified, they can be furtheroptimized by adjusting sequence, structure, modification, etc., toachieve desired characteristics, such as DM and PK.

Characteristics of iRNA include, but are not limited to, stability,efficacy, and toxicity.

The iRNA may also be optimized in conjunction with a delivery system asprovided herein, or known in the art.

III. Method of Treatments

In another aspect, the present invention provided a method for treatinga disease, comprising administering a pharmaceutically effective amountof the iRNA provided herein to a subject in need of such treatment.

The term “subject,” or “individual” as used herein in reference toindividuals suffering from a disorder, and the like, encompasses mammalsand non-mammals. Examples of mammals include, but are not limited to,any member of the Mammalian class: humans, non-human primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, horses, sheep, goats, swine; domestic animals such as rabbits,dogs, and cats; laboratory animals including rodents, such as rats, miceand guinea pigs, and the like. Examples of non-mammals include, but arenot limited to, birds, fish and the like. In some embodiments of themethods and compositions provided herein, the mammal is a human.

The RNA molecule of this invention can be administered in conjunctionwith RNA transfer vector and other known treatments for a diseasecondition.

The associated formulations of drugs vary in compliance with theadministration of the corresponding diseases and are appropriate tomaintain the activity of iRNA molecules. For example, for injectabledrug together with proper delivery systems, the formulation can be alyophilized powder.

Optionally, the above drug formulations can contain any pharmaceuticalacceptable adjuvant, as long as the appropriate delivery systemssuitable and appropriate to maintain the activity of RNA molecules.

The iRNA molecule of the present invention may be administered in anyform, for example transdermally or by local injection.

The compositions of the present invention may also be formulated andused as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions, suspensionsfor injectable administration, and the other forms known in the art.

In a preferred embodiment, the iRNA comprised of an antisense and asense strand, the sense and antisense strand annealed into duplex, theduplex contains a non-complementary loop structure. The antisense strandtarget two and/or more target RNA of genes or two and/or more sites ofone target RNA of a target gene, and it does not induce aninterferon-response when introduced (e.g. y transfection) into a cell.

IV. Pharmaceutical Compositions and Delivery

In yet another aspect, the present invention provides a pharmaceuticalcomposition, comprising the iRNA of provided herein, and apharmaceutical acceptable carrier.

The present invention provides the preparation of iRNA molecules totreat pathological angiogenesis-related diseases. The associatedformulations of drugs vary in compliance with the administration of thecorresponding diseases and are appropriate to maintain the activity ofiRNA molecules. For example, for injectable drug together with properdelivery systems, the formulation can be a lyophilized powder.

Optionally, the above drug formulations can contain any pharmaceuticalacceptable adjuvant, as long as the appropriate delivery systemssuitable and appropriate to maintain the activity of iRNA molecules.

For example, in clinical application of ophthalmic drugs, the iRNA ofthe present invention can be dissolved in sterile water free of RNAenzymes. The iRNA concentration is adjusted to 1 μg/μL. The intravitrealinjection is performed after gentle mixture of the preparation. Theinjection is conducted once every two weeks, 4 weeks as a course of atreatment.

In another preferred embodiment, treatment of a patient comprisesadministration one or more of the RNA compounds, in conjunction withother therapies, for example, chemotherapy, radiation, surgery,anti-inflammatory agents and the like. The other agents can beadministered, prior to, after or co-administered with the RNA compounds.

The RNA compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal comprising a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bio-equivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

As used herein, a “pharmaceutical addition salt” comprises apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These comprise organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and comprise basic salts of a variety of inorganicand organic acids, such as, for example, with inorganic acids, such asfor example hydrochloric acid, hydrobromic acid, sulfuric acid orphosphoric acid; with organic carboxylic, sulfonic, sulfo or phosphoacids or N-substituted sulfamic acids, for example acetic acid,propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleicacid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lacticacid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin Nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and comprise alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.For oligonucleotides, preferred examples of pharmaceutically acceptablesalts comprise but are not limited to: (I) salts formed with cationssuch as sodium, potassium, ammonium, magnesium, calcium, polyamides suchas spermine and spermidine, and the like; (II) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; (III)salts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and(IV) salts formed from elemental anions such as chlorine, bromine, andiodine.

The RNA compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of a target gene is treated by administering RNA compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan iRNA compound to a suitable pharmaceutically acceptable diluent orcarrier. Use of the RNA compounds and methods of the invention may alsobe useful prophylactically.

The present invention also comprises pharmaceutical compositions andformulations, which comprise the RNA compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (comprising ophthalmic and to mucous membranes comprisingvaginal and rectal delivery), pulmonary, e.g., by inhalation of powdersor aerosols, comprising by nebulizer, intratracheal, intranasal,epidermal and transdermal), oral or parenteral.

Pharmaceutical compositions of the present invention comprise, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that comprise, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is co-administered with polyinosinic acid, dextran sulphate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more RNA compounds and one or more otherchemotherapeutic agents which function by a non-TRL related mechanism.

Examples of such chemotherapeutic agents comprise, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MIX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228).

Anti-inflammatory drugs, comprising but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,comprising but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention (TheMerck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds.,1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Othernon-antisense chemotherapeutic agents are also within the scope of thisinvention. Two or more combined compounds may be used together orsequentially.

In another related embodiment, compositions of the invention may containone or more RNA compounds, particularly iRNAs with different sequences.Two or more combined compounds may be used together or sequentially.

Nucleic Acid Delivery System

Preferred invention practice involves administering at least one of theforegoing RNA oligonucleotides with a suitable nucleic acid deliverysystem, e.g. as disclosed in US Pat. Appl. Pub. No. 20090247604, thedisclosure of which are incorporated by reference in its entirety.

In one embodiment, that system includes a non-viral vector operablylinked to the polynucleotide. Examples of such non-viral vectors includethe oligonucleotide alone or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutination virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally preferred vectors include viral vectors, fusion proteinsand chemical conjugates. Retroviral vectors include Moloney murineleukemia viruses and HIV-based viruses. One preferred HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors are preferred. These vectors include pox vectors such asorthopox or avipox vectors, herpesvirus vectors such as a herpes simplexI virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487(1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover,Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al.,Proc Natl. Acad. Sci.: U.S.A.: 90 7603 (1993); Geller, A. I., et al.,Proc Natl. Acad. Sci USA: 87:1149 (1990)], Adenovirus Vectors [LeGalLaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet.3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995)] andAdeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet.8:148 (1994)].

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors may be an indication for some inventionembodiments. The adenovirus vector results in a shorter term expression(e.g., less than about a month) than adeno-associated virus, in someembodiments, may exhibit much longer expression. The particular vectorchosen will depend upon the target cell and the condition being treated.

The selection of appropriate promoters can readily be accomplished.Preferably, one would use a high expression promoter. An example of asuitable promoter is the 763-base-pair cytomegalovirus (CMV) promoter.The Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther 4:151 (1993))and MMT promoters may also be used. Certain proteins can be expressedusing their native promoter. Other elements that can enhance expressioncan also be included such as an enhancer or a system that results inhigh levels of expression such as a tat gene and tar element. Thiscassette can then be inserted into a vector, e.g., a plasmid vector suchas, pUC19, pUC118, pBR322, or other known plasmid vectors, thatincludes, for example, an E. coli origin of replication. See, Sambrook,et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory press, (1989). Promoters are discussed infra. The plasmidvector may also include a selectable marker such as the β-lactamase genefor ampicillin resistance, provided that the marker polypeptide does notadversely effect the metabolism of the organism being treated. Thecassette can also be bound to a nucleic acid binding moiety in asynthetic delivery system, such as the system disclosed in WO 95/22618.

If desired, the polynucleotides of the invention may also be used with amicrodelivery vehicle such as cationic liposomes and adenoviral vectors.For a review of the procedures for liposome preparation, targeting anddelivery of contents, see Mannino and Gould-Fogerite, BioTechniques,6:682 (1988). See also, Felgner and Holm, Bethesda Res. Lab. Focus,11(2):21 (1989) and Maurer, R. A., Bethesda Res. Lab. Focus, 11(2):25(1989).

Replication-defective recombinant adenoviral vectors can be produced inaccordance with known techniques. See, Quantin, et al., Proc. Natl.Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J.Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell,68:143-155 (1992).

Another preferred antisense oligonucleotide delivery method is to usesingle stranded DNA producing vectors which can produce the antisenseoligonucleotides intracellularly. See for example, Chen et al,BioTechniques, 34: 167-171 (2003), which is incorporated herein, byreference, in its entirety.

The effective dose of the nucleic acid will be a function of theparticular expressed protein, the particular cardiac arrhythmia to betargeted, the patient and his or her clinical condition, weight, age,sex, etc.

One preferred delivery system is a recombinant viral vector thatincorporates one or more of the polynucleotides therein, preferablyabout one polynucleotide. Preferably, the viral vector used in theinvention methods has a pfu (plague forming units) of from about 10⁸ toabout 5×10¹⁰ pfu. In embodiments in which the polynucleotide is to beadministered with a non-viral vector, use of between from about 0.1nanograms to about 4000 micrograms will often be useful e.g., about 1nanogram to about 100 micrograms.

Embodiments of the invention also relates to expression vectorconstructs for the expression of the RNA oligonucleotides which containhybrid promoter gene sequences and possess a strong constitutivepromoter activity or a promoter activity which can be induced in thedesired case.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor gene products in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding genes of interest.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for polymerases. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. Promoters are composed of discrete functional modules, eachconsisting of approximately 7-20 bp of DNA, and containing one or morerecognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 b.p.upstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 b.p. apart before activitybegins to decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constitutively expressed fromone vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.). This system also allows highlevels of gene expression to be regulated in response to tetracycline ortetracycline derivatives such as doxycycline. In the Tet-On™ system,gene expression is turned on in the presence of doxycycline, whereas inthe Tet-Off™ system, gene expression is turned on in the absence ofdoxycycline. These systems are based on two regulatory elements derivedfrom the tetracycline resistance operon of E. coli. The tetracyclineoperator sequence to which the tetracycline repressor binds, and thetetracycline repressor protein. The gene of interest is cloned into aplasmid behind a promoter that has tetracycline-responsive elementspresent in it. A second plasmid contains a regulatory element called thetetracycline-controlled transactivator, which is composed, in theTet-Off™ system, of the VP16 domain from the herpes simplex virus andthe wild-type tetracycline repressor. Thus in the absence ofdoxycycline, transcription is constitutively on. In the Tet-On™ system,the tetracycline repressor is not wild type and in the presence ofdoxycycline activates transcription. For gene therapy vector production,the Tet-Off™ system would be preferable so that the producer cells couldbe grown in the presence of tetracycline or doxycycline and preventexpression of a potentially toxic transgene, but when the vector isintroduced to the patient, the gene expression would be constitutivelyon.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic Virus, HSV-TK, and avian sarcoma virus.

In a preferred embodiment, tissue specific promoters are used to effecttranscription in specific tissues or cells so as to reduce potentialtoxicity or undesirable effects to non-targeted tissues. For example,promoters such as the PSA, probasin, prostatic acid phosphatase orprostate-specific glandular kallikrein (hK2) may be used to target geneexpression in the prostate.

IRES:

In certain embodiments of the invention, the use of internal ribosomeentry site (IRES) elements is contemplated to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites. IRES elements from two members of thepicornavirus family (poliovirus and encephalomyocarditis) have beendescribed, as well an IRES from a mammalian message. IRES elements canbe linked to heterologous open reading frames. Multiple open readingframes can be transcribed together, each separated by an IRES, creatingpolycistronic messages. By virtue of the IRES element, each open readingframe is accessible to ribosomes for efficient translation. Multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

Kits

In another aspect, the present invention provides a kit comprises one ormore RNA oligonucleotides provided herein. These oligonucleotides cancomprise one or more modified nucleobases, shorter or longer fragments,modified bonds and the like. In yet another aspect, the inventionprovides kits for targeting nucleic acid sequences of cells andmolecules associated with modulation of the immune response in thetreatment of diseases such as, for example, infectious diseaseorganisms, AMD, angiogenesis related diseases, cancer, autoimmunediseases and the like.

In one embodiment, a kit comprises: (a) an RNA provided herein, and (b)instructions to administer to cells or an individual a therapeuticallyeffective amount of RNA oligonucleotide. In some embodiments, the kitmay comprise pharmaceutically acceptable salts or solutions foradministering the RNA oligonucleotide. Optionally, the kit can furthercomprise instructions for suitable operational parameters in the form ofa label or a separate insert. For example, the kit may have standardinstructions informing a physician or laboratory technician to prepare adose of RNA oligonucleotide.

Optionally, the kit may further comprise a standard or controlinformation so that a patient sample can be compared with the controlinformation standard to determine if the test amount of RNAoligonucleotide is a therapeutic amount consistent with for example, ashrinking of a tumor.

Embodiments of the invention may be practiced without the theoreticalaspects presented. Moreover, the theoretical aspects are presented withthe understanding that Applicants do not seek to be bound by the theorypresented.

EXAMPLES

It should be understood that the following examples used only to clarifythe present invention, but not to limit this invention.

It must be explained, if not specified, that the percentage of followingexamples are all weight percent content wt %.

Example 1

Design of iRNA Molecule Mid-Cir Targeting Survivin and Bcl-2

Cell apoptosis suppressor gene Survivin and Bcl-2 are high expressionwidely in suppressor gene. The study found that, tumor cells growth canbe suppressed through the inhibition of Survivin or Bcl-2 geneover-expression in tumor cells. The targeted siRNA can be used forcancer therapy through Survivin or Bcl-2 gene expression inhibition.

Design of iRNA molecule Mid-Cir, which contains a sense and antisensestrand, the 3′ segment of antisense strand target an mRNA that encodesSurvivin gene, the 5′ segment of antisense strand target an mRNA thatencodes Bcl-2 gene, and the structure Mid-Cir of shown in FIG. 1.

SEQ ID NO: 1 shows a sense sequence of an iRNA Mid-Cir.

Sense strand: (SEQ ID NO: 1)5′-GACUUGGCCCAGUGUUUCUUCAGGAUGACUGAGUACCUGAA-3′.

SEQ ID NO: 2 is an antisense sequence that complementary to a sensesequence of an iRNA Mid-Cir with non-complementary loop structure.

Antisense strand: (SEQ ID NO: 2)5′-UUCAGGUACUCAGUCAUCCGAGAGAAACACUGGGCCAAGUC-3′.

The control siRNAs: 1) Sur-2 is the siRNA that target Survivin gene, thesense sequence is the 1˜19 nucleotides from 5′end of SEQ ID NO: 1,antisense sequence is the 1˜19 nucleotides from 3′end of SEQ ID NO: 1.2) Bcl-1 is the siRNA that target Bcl-2 gene, the sense sequence is the1˜19 nucleotides from 3′end of SEQ ID NO: 2, antisense sequence is the1˜19 nucleotides from 5′end of SEQ ID NO: 2.

Example 2

Design of iRNA Molecule Mid-Loop Targeting Survivin and Bcl-2

Cell apoptosis suppressor gene Survivin and Bcl-2 are high expressionwidely in suppressor gene. The study found that, tumor cells growth canbe suppressed through the inhibition of Survivin or Bcl-2 geneover-expression in tumor cells. The targeted siRNA can be used forcancer therapy through Survivin or Bcl-2 gene expression inhibition.

Design of iRNA molecule Mid-Loop, which contains a sense and antisensestrand, the 3′ segment of antisense strand target an mRNA that encodesSurvivin gene, the 5′ segment of antisense strand target an mRNA thatencodes Bcl-2 gene, and the structure of Mid-Loop shown in FIG. 2.

SEQ ID NO: 3 shows a sense sequence of an iRNA Mid-Loop.

Sense strand: (SEQ ID NO: 3)5′-GACUUGGCCCAGUGUUUCUCAUGCGUCGGGAUGACUGAGUACCUG AA-3′.

SEQ ID NO: 4 is an antisense sequence that complementary to a sensesequence of an iRNA Mid-Loop with self-complementary looped nucleotidehairpin structure.

Antisense strand: (SEQ ID NO: 4)5′-UUCAGGUACUCAGUCAUCCGCAGCACACAGAAACACUGGGCCAAG UC-3′.

The control siRNAs: 1) Sur-2 is the siRNA that target Survivin gene, thesense sequence is the 1˜19 nucleotides from 5′end of SEQ ID NO: 1,antisense sequence is the 1˜19 nucleotides from 3′end of SEQ ID NO: 1.2) Bcl-1 is the siRNA that target Bcl-2 gene, the sense sequence is the1˜19 nucleotides from 3′end of SEQ ID NO: 2, antisense sequence is the1˜19 nucleotides from 5′end of SEQ ID NO: 2.

Example 3

Design of iRNA Molecule By-Pass Targeting Survivin and Bcl-2

Cell apoptosis suppressor gene Survivin and Bcl-2 are high expressionwidely in suppressor gene. The study found that, tumor cells growth canbe suppressed through the inhibition of Survivin or Bcl-2 geneover-expression in tumor cells. The targeted siRNA can be used forcancer therapy through Survivin or Bcl-2 gene expression inhibition.

Design of iRNA molecule By-Pass, which contains a sense and twoantisense strand: antisense strand 1 and antisense strand 2, the 3′ and5′ segment of antisense strand 1 target an mRNA that encodes Survivingene, the 5′ segment of antisense strand 2 target an mRNA that encodesBcl-2 gene, and the 3′ segment of antisense strand 2 target an mRNA thatencodes Bcl-2 gene, and the structure of By-Pass shown in FIG. 3.

SEQ ID NO: 5 shows a sense sequence of an iRNA By-Pass.

Sense strand: (SEQ ID NO: 5)5′-GACUUGGCCCAGUGUUUCUCAUGCGUCGGGAUGACUGAGUACCUG AA-3′.

SEQ ID NO: 6 is the antisense strand 1 that 3′ segment is complementaryto the 5′ segment of SEQ ID NO: 5, SEQ ID NO: 7 is the antisense strand2 that 5′ segment is complementary to the 3′ segment of SEQ ID NO: 5,the 5′ segment of SEQ ID NO: 6 is complementary to the 5′ segment of SEQID NO: 7.

Antisense strand 1: (SEQ ID NO: 6)5′-UCCUUUCUGUCAAGAAGCAGUUCAGAAACACUGGGCCAAGUC-3′. Antisense strand 2:(SEQ ID NO: 7) 5′-UUCAGGUACUCAGUCAUCCCAACUGCUUCUUGACAGAAAGGA-3′.

The control siRNAs: 1) Sur-2 is the siRNA that target Survivin gene, thesense sequence is the 1˜19 nucleotides from 5′end of SEQ ID NO: 1,antisense sequence is the 1˜19 nucleotides from 3′end of SEQ ID NO: 1.2) Bcl-1 is the siRNA that target Bcl-2 gene, the sense sequence is the1˜19 nucleotides from 3′end of SEQ ID NO: 2, antisense sequence is the1˜19 nucleotides from 5′end of SEQ ID NO: 2.

Example 4

Real-Time Quantitative PCR (RT-qPCR) Detection the Silencing Effects ofSurvivin Target Gene by iRNA Molecules Mid-Cir, Mid-Loop, By-Pass.

Cell culture: SMMC-7721 cells are cultured in DMEM supplemented with 10%FBS (Gibco Inc.) at 37° C. supplied with 5% CO2.

Cell Plated and transfection: 1×105 cells/well were plated in a 96-wellplate and grown in DMEM supplemented with 10% FBS (Gibco Inc.) at 37° C.supplied with 5% CO2 overnight. The procedure of transfection followedby Lipofectamin™2000 (Invitrogen Inc.) protocol. The concentrations ofexperimental RNA molecules were 10 nM/well.

The level of mRNA encoding Survivin was determined by RT-qPCR: the cellmRNAs were extracted by TurboCapture mRNA Kit (QIAGEN Inc.), theprocedure followed by the protocol. 80 μl RNase free water was added todissolve the RNA, and 4 μl RNA was took as template to RT-qPCRamplification.

The primers used to detect the level of Survivin gene by RT-qPCR were:

5′ Forward primer: (SEQ ID NO: 8) ACCGCATCTCTACATTCAAG, 3′Reverse primer: (SEQ ID NO: 9) CAAGTCTGGCTCGTTCTC.

The level of mRNA encoding Survivin was determined by RT-qPCR, meanwhileGAPDH was determined as the loading control, and 3 repeat reactions wereset up per sample. The 25 μl reaction mix contained: 4 μl template RNA,12.5 μl of 2×SensiMix One-Step (Quantance), 1 μl 5′ forward and 3′reverse primer (10 μM), 0.5 μl 50×SYBR Green I and added RNase freewater to 25 μl. The reaction was repeated for 45 cycles as reversetranscription at 42° C. for 30 min, preheating at 95° C. for 7 min,denaturing at 95° C. for 20 s, annealing at 60° C. for 30 s, andextension at 72° C. for 30 s.

As shown in FIG. 4, the results were analyzed by 2-ΔΔCt methods ofhistogram. The iRNA molecules Mid-Cir, Mid-Loop, By-Pass all had shownthe better results than the corresponding Sur-2 transfection group andSur-2/Bcl-1 co-transfection group in the Survivin gene silencing.

Example 5

RT-qPCR Detection the Silencing Effects of Bcl-2 Target Gene by iRNAMolecules Mid-Cir, Mid-Loop, By-Pass.

Cell culture: SMMC-7721 cells are cultured in DMEM supplemented with 10%FBS (Gibco Inc.) at 37° C. supplied with 5% CO2.

Cell Plated and transfection: 1×105 cells/well were plated in a 96-wellplate and grown in DMEM supplemented with 10% FBS (Gibco Inc.) at 37° C.supplied with 5% CO2 overnight. The procedure of transfection followedby Lipofectamin™2000 (Invitrogen Inc.) protocol. The concentrations ofexperimental RNA molecules were 10 nM/well.

The level of mRNA encoding Bcl-2 was determined by RT-qPCR: the cellmRNAs were extracted by TurboCapture mRNA Kit (QIAGEN Inc.), theprocedure followed by the protocol. 80 μl RNase free water was added todissolve the RNA, and 4 μl RNA was took as template to RT-qPCRamplification.

The primers used to detect the level of Bcl-2 gene by RT-qPCR were:

5′ Forward primer: (SEQ ID NO: 10) GGCTGGGATGCCTTTGTG, 3′Reverse primer: (SEQ ID NO: 11) GCCAGGAGAAATCAAACAGAGG.

The level of mRNA encoding Bcl-2 was determined by RT-qPCR, meanwhileGAPDH was determined as the loading control, and 3 repeat reactions wereset up per sample. The 25 μL reaction mix contained: 4 μl template RNA,12.5 μl of 2×SensiMix One-Step (Quantance), 1 μl 5′ forward and 3′reverse primer (10 μM), 0.5 μl 50×SYBR Green I and added RNase freewater to 25 μl. The reaction was repeated for 45 cycles as reversetranscription at 42° C. for 30 min, preheating at 95° C. for 7 min,denaturing at 95° C. for 20 s, annealing at 60° C. for 30 s, andextension at 72° C. for 30 s.

As shown in FIG. 5, the results were analyzed by 2-ΔΔCt methods ofhistogram. The iRNA molecules Mid-Cir, Mid-Loop, By-Pass all had shownthe better results than the corresponding Bcl-1 transfection group andSur-2/Bcl-1 co-transfection group in the Bcl-2 gene silencing.

Example 6

Interferon Response was Measured by RT-qPCR.

Cell culture: SMMC-7721 cells are cultured in DMEM supplemented with 10%FBS (Gibco Inc.) at 37° C. supplied with 5% CO2.

Cell Plated and transfection: 1×105 cells/well were plated in a 96-wellplate and grown in DMEM supplemented with 10% FBS (Gibco Inc.) at 37° C.supplied with 5% CO2 overnight. The procedure of transfection followedby Lipofectamin™2000 (Invitrogen Inc.) protocol. The concentrations ofexperimental RNA molecules were 10 nM/well.

The level of mRNA encoding interferon-related gene OAS1 was determinedby RT-qPCR: the cell mRNAs were extracted by TurboCapture mRNA Kit(QIAGEN Inc.), the procedure followed by the protocol. 80 μl RNase freewater was added to dissolve the RNA, and 4 μl RNA was took as templateto RT-qPCR amplification.

The primers used to detect the level of interferon-related gene OAS1 byRT-qPCR were:

5′ Forward primer: (SEQ ID NO: 12) GTGAGCTCCTGGATTCTGCT, 3′Reverse primer: (SEQ ID NO: 13) TGTTCCAATGTAACCATATTTCTGA.

The level of mRNA encoding OAS1 was determined by RT-qPCR, meanwhileGAPDH was determined as the loading control, and 3 repeat reactions wereset up per sample. The 25 μL reaction mix contained: 4 μl template RNA,12.5 μl of 2×SensiMix One-Step (Quantance), 1 μl 5′ forward and 3′reverse primer (10 μM), 0.5 μl 50×SYBR Green I and added RNase freewater to 25 μl. The reaction was repeated for 45 cycles as reversetranscription at 42° C. for 30 min, preheating at 95° C. for 7 min,denaturing at 95° C. for 20 s, annealing at 60° C. for 30 s, andextension at 72° C. for 30 s.

As shown in FIG. 6, the results were analyzed by 2-ΔΔCt methods ofhistogram. During experiment, 5 ng/well Polyinosine-polycytidylic acid(Poly(I:C)) were transfected as positive control. Poly(I:C) is asynthetic analog of double-stranded RNA (dsRNA), a molecule patternassociated with viral infection, and it can induce antimicrobial immuneresponses. Comparisons with the normal group, the mRNA of OAS1 gene inpositive control group were step-up significantly; the OAS1 gene ofother group had no high expression. The results indicated the iRNAmolecules Mid-Cir, Mid-Loop, By-Pass had not induced the interferonresponse.

Example 7

The Mid-Cir, Mid-Loop, By-Pass Inhibition Rates of SMMC-7721 Cells wereDetected by CCK-8.

Cell culture: SMMC-7721 cells are cultured in DMEM supplemented with 10%FBS (Gibco Inc.) at 37° C. supplied with 5% CO2.

Cell Plated and transfection: 1×105 cells/well were plated in a 96-wellplate and grown in DMEM supplemented with 10% FBS (Gibco Inc.) at 37° C.supplied with 5% CO2 overnight. The procedure of transfection followedby Lipofectamin™2000 (Invitrogen Inc.) protocol. The concentrations ofexperimental RNA molecules were 10 nM/well.

CCK-8 detection: 1/10 volume CCK-8 solution (Dojindo) of medium wereadded per well. Then, cells were keep incubation in cell culture ovenfor 0.5˜4 h. The absorbance values were measured at 450 nm wave-lengthby microplate reader (Bio-Rad).

As shown in FIG. 7, the growth curve was made according to the A450values. The histogram was shown in FIG. 8, the cell growth inhibitionrates were calculated according to the formula: cell proliferationrate=(1−A450 mean value per group/A450 mean value of controlgroup)×100%. The results show that the hepatoma cells were inhibitedsignificantly after the treatment of the iRNA molecules Mid-Cir,Mid-Loop, By-Pass for 48 hrs, 72 hrs, 96 hrs.

Example 8

The Mid-Cir, Mid-Loop, By-Pass Inhibition Rates of HepG2 Cells wereDetected by CCK-8.

Cell culture: HepG2 cells are cultured in DMEM supplemented with 10% FBS(Gibco Inc.) at 37° C. supplied with 5% CO2.

Cell Plated and transfection: 1×105 cells/well were plated in a 96-wellplate and grown in DMEM supplemented with 10% FBS (Gibco Inc.) at 37° C.supplied with 5% CO2 overnight. The procedure of transfection followedby Lipofectamin™2000 (Invitrogen Inc.) protocol. The concentrations ofexperimental RNA molecules were 10 nM/well.

CCK-8 detection: 1/10 volume CCK-8 solution (Dojindo) of medium wereadded per well. Then, cells were keep incubation in cell culture ovenfor 0.5˜4 h. The absorbance values were measured at 450 nm wave-lengthby microplate reader (Bio-Rad).

As shown in FIG. 9, the growth curve was made according to the A450values. The histogram was shown in FIG. 10, the cell growth inhibitionrates were calculated according to the formula: cell proliferationrate=(1−A450 mean value per group/A450 mean value of controlgroup)×100%. The results show that the hepatoma cells were inhibitedsignificantly after the treatment of the iRNA molecules Mid-Cir,Mid-Loop, By-Pass for 48 hrs, 72 hrs, 96 hrs.

Example 9

In Vivo Gene Silencing Analysis

All procedures used in animal studies conducted at Alnylam are approvedby the Institutional Animal Care and Use Committee (IACUC). The effectsof the multi-targets iRNA molecules are validated in the mice bearingliver cancer via intratumoral injection. Liver tissue was dissolved inproteinase K-containing cell and tissue lysis buffer (Sigma) andsubjected to tissue lysis with high shear forces. Total RNA wasextracted with Qiagen RNA extraction kit. The expression levels of Bcl-2and Survivin were determined by RT-qPCR. And the tumor inhibition rateis evaluated by the volumes of tumor and etc.

What is claimed is:
 1. An interfering RNA (iRNA) molecule, comprising: asense strand and an antisense strand annealed to said sense strand,wherein the sense strand and the antisense strand comprise the sequencesof SEQ ID NOs: 3 and 4, respectively.
 2. The iRNA of claim 1, whereinsaid iRNA comprises a sugar, base, or phosphate modification, or acombination thereof.
 3. A pharmaceutical composition comprising the iRNAof claim 1, and a pharmaceutical acceptable excipient.
 4. An interferingRNA (iRNA) molecule, comprising three non-continuous strands: a sensestrand, a first antisense strand, and a second antisense strand, whereinthe sense strand, the first antisense strand, and the second antisensestrand comprise the sequences of SEQ ID NOs: 5-7, respectively.
 5. Apharmaceutical composition comprising the iRNA of claim 4, and apharmaceutical acceptable excipient.
 6. The iRNA of claim 4, whereinsaid iRNA comprises a sugar, base, or phosphate modification, or acombination thereof.